16S rRNA gene amplicon sequencing of cutaneous swabs

For 16S rRNA gene sequencing, WT mice were analyzed at the indicated time points without any EGFR^Δep mice present in the litter to avoid contamination with the microbiome of the mutant mice. Eurotubo Collection swabs (Deltalab) were prewetted in 1 ml of sterile PBS, and a defined area of dorsal skin was sampled (vigorously swabbed for 10 s). Negative control swabs were prewetted and exposed to the room environment. The QIAamp DNA Microbiome Kit (QIAGEN), which includes a step for depletion of host cell DNA, was used for DNA extraction according to the manufacturer’s guidelines.

Amplicon sequencing of 16S rRNA genes was performed as described previously (42). Sequences were amplified and barcoded using a two-step polymerase chain reaction (PCR) approach. In the first step, 16S rRNA genes were amplified using degenerate primers that target most bacteria and archaea (H_341F 5′-GCTATGCGCGAGCTGCCCTACGGGNGGCWGCAG and H_785R 5′-GCTATGCGCGAGCTGCGACTACHVGGGTATCTAATCC; both primers contain a universal “HEAD” sequence as target for a barcoding primer in the second PCR) (42). Blank nucleic acid extractions and negative (water only) PCRs were included as controls. All first-step PCRs were prepared in triplicate (20-μl volume) containing 1× DreamTaq buffer (Thermo Scientific), 0.2 mM deoxynucleotide triphosphate mix (Thermo Scientific), 1-U DreamTaq (Thermo Scientific), BSA (0.2 mg/ml) (Thermo Scientific), 1 μM each forward and reverse primer mix, and 1 μl of template. Thermal cycling conditions were 95°C for 3 min; 25 cycles of 95°C for 30 s, 52°C for 30 s, 72°C for 1 min; final extension at 72°C for 7 min. After confirmation of product formation by gel electrophoresis, replicate PCRs were pooled and cleaned using the Zymogen DNA Clean and Concentrator kit (Zymo Research Corp.). Amplicons were eluted in 30 μl of nuclease-free water. Second-step barcoding PCRs (50-μl volume) contained 1 μl of the cleaned first-step PCR product as template and were subjected to thermal cycling conditions of 95°C for 3 min; 5 cycles of 95°C for 30 s, 52°C for 30 s, 72°C for 1 min; final extension at 72°C for 7 min. PCR products were again checked by gel electrophoresis and cleaned as previously described. DNA was quantified using Quant-iT PicoGreen double-stranded DNA (dsDNA) kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Barcoded amplicons from different samples were pooled at equivalent copy numbers (2 × 10¹⁰) and paired-end sequenced (Microsynth AG) on an Illumina MiSeq sequencer system [2 x 300 base pairs (bp)]. Sequencing results were processed into library-specific paired-end reads as outlined previously (42). Paired-end reads were assembled using fastq-join, clustered into chimera-filtered operational taxonomic units (OTUs) (97% identity) with UPARSE, and taxonomic classification was determined with the Ribosomal Database Project (RDP) classifier implemented in Mothur.

16S rRNA gene amplicon sequence variants (ASVs) at single-nucleotide resolution were determined from paired-end reads using dada2 with the filterAndTrim (truncQ = 2, minLen = 250), learnErrors(), derepFastq(), dada(), and mergePairs() functions, and taxonomic classification was determined with the RDP classifier implemented in Mothur. ASVs classified at the genus level (>80% confidence) as Staphylococcus were placed into a reference tree of Staphylococcus strains using RAxML-EPA. The reference tree was constructed using RAxML from 74 type strain 16S rRNA sequences obtained from the RDP and aligned with the SINA aligner. The presence of contaminant sequences particularly afflicting low-biomass samples is well documented (43, 44). Therefore, a conservative curation of identified OTUs was performed. First, we retained OTUs with greater than 10 read counts in any one sample. Second, OTUs with greater read counts in negative controls (DNA extraction and PCR controls) were removed. Third, to account for potential cross contamination occurring during sequencing, OTUs in other barcoded samples with read counts three orders of magnitude higher were examined as possible candidate cross-contaminating sequences.

For alpha-diversity analyses, ASV tables were imported into the R software environment (R Core Team, 2015) and processed within the package phyloseq (45). The datasets were subsampled at the depth of the smallest library. Rarefied ASV tables were then used to calculate alpha-diversity indices (Shannon diversity, Simpson, and inverse Simpson).

Species (for example, Staphylococcus-type species) within a species group (S. aureus/Staphylococcus argenteus versus S. xylosus/ Staphylococcus saprophyticus) had identical 16S rRNA gene sequences and, thus, could not be distinguished by 16S rRNA sequencing. Therefore, parallel cultivation-based analyses using Staphylococcus identification plates (SAID, bioMérieux) revealed the identity of the species within each species group.